FR2786700A1 - Agent for transferring nucleic acid, useful e.g. for gene therapy, contains hydrophobic spacer linking polycation and hydrophilic substituents - Google Patents

Abstract

Agent (I) for transfer of nucleic acid (II) comprises a hydrophobic spacer (Sp) chemically linked to (i) a polycation (PC) and (ii) at least one hydrophilic substituent (HS). An Independent claim is also included for a composition comprising (I) and (II).

Description

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NOVEL NUCLEIC ACID TRANSFER AGENTS,
COMPOSITIONS CONTAINING SAME AND USES THEREOF
The present invention relates to novel transfer agents, compositions containing them and their uses for the in vitro, in vivo or ex vivo transfer of nucleic acids into cells.

 With the development of biotechnologies, the ability to efficiently transfer nucleic acids into cells has become a basic technique with many biotechnological applications. It can be the transfer of nucleic acids in cells in vitro, for example for the production of recombinant proteins, or in the laboratory for the study of the regulation of gene expression, the cloning of genes or any other manipulation involving DNA. It may also be the transfer of nucleic acids into cells in vivo, for example for the production of vaccines, labeling studies or also therapeutic approaches. It can also be the transfer of genes into cells taken from an organism, for later readministration, for example for the creation of transgenic animals.

 Currently, the most common way to transfer genes into cells is the use of viral vectors. But since these are not completely harmless, several other methods based on the use of synthetic vectors have been proposed. These synthetic vectors have two main functions: complexing and compacting the nucleic acid to be transfected, and promoting its passage through the plasma membrane and possibly through the two nuclear membranes.

 Several families of synthetic vectors have been developed, such as polymers or biochemical vectors (consisting of a cationic protein associated with a cellular receptor), but significant progress has been made above all in non-viral transfection with the development of lipofectants, and more particularly cationic lipids. It has thus been demonstrated that lipids

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 Cationic, because of their overall positive charge, spontaneously interfered with the overall negative DNA, forming nucleolipid complexes capable of fusing with cell membranes, and thus allowed the intracellular release of DNA.

 Different kinds of cationic lipids have thus been synthesized: lipids comprising a quaternary ammonium group (for example DOTMA, DOTAP, DMRIE, DLRIE, etc.), lipopolyamines such as for example DOGS, DC-Chol or the lipopolyamines disclosed. in the patent application WO 97/18185, lipids combining both a quaternary ammonium group and a polyamine such as DOSPA, or lipids comprising various other cationic entities, especially amidinium groups (for example ADPDE, ADODE or the lipids of the patent application WO 97/31935). In fact, the structural diversity of cationic lipids partly reflects the observation of the structure-activity relationship.

 However, the use of these synthetic vectors still poses many difficulties, and their effectiveness remains to be improved. In particular, it would be desirable to have non-cationic or less cationic vectors, for two main reasons. The first is that the complexes formed between the nucleic acid and the transfer vectors, because of their overall positive charge, tend to accumulate in the vesiculoendothelial system of the cells and thus cause an inflammatory response. The second reason is a question of improving the bioavailability, that is to say the ability of the complexes to reach the target cells to be transfected. Indeed, because of the overall positive charge of the complexes formed, the plasma proteins tend to adsorb to their surface. the nucleic acid complexes can therefore no longer reach the target cells, which consequently leads to a decrease in the transfer efficiency with respect to the quantity of complexes injected.

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 Moreover, the stable formulation of synthetic vectors developed to date at low charge ratios is generally difficult or impossible.

In the following, "charge ratio" means the ratio of the positive charges of the transfer agent to the negative charges of the DNA. This ratio is often expressed in nmoles of transfer agent per g of DNA.

 These are the problems that the new transfecting agents developed by the Applicant, and which are the subject of the present invention, propose to solve.

Indeed, their particular structure which forms a hydrophobic anchor with two hydrophilic heads, at least one of which is a sugar, makes it possible to reduce the charge density of these vectors with respect to the cationic lipids known to those skilled in the art, because these structures form a sort of "screen of charges". The bioavailability of the complexes formed with the nucleic acids is therefore improved, since this results in a decrease in the adsorption of the plasma proteins on their surface. In addition, "masking" the charges represents a significant improvement in toxicity. Finally, the transfecting agents of the present invention are particularly advantageous from a physico-chemical point of view. Indeed, the applicant has demonstrated, quite surprisingly and unexpectedly, that the transfer agents according to the invention are particularly stable when they are brought into contact with nucleic acids at low charge ratios.

 Thus, a first subject of the invention relates to novel nucleic acid transfer agents which comprise a hydrophobic part bonded to at least two hydrophilic parts, at least one of which is a sugar.

More particularly, the transfer agents according to the invention are represented by the general formula (I).

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 for which: R represents a polycation; Z represents a hydrogen atom or a fluorine atom, the different Zs being independent of one another; and x and y, independently of one another, represent an integer; between 10 and 22 inclusive, and - X and Y, independently of each other, represent a hydrogen atom, an OAlk group, Alk representing a straight or branched alkyl containing 1 to 4 carbon atoms, a group amino, a sugar, a peptide, an oligonucleotide or a marker, at least one of X and Y substituents being a sugar.

 For the purposes of the invention, the term "sugar" means any molecule of mono-, oligo or polysaccharide which interacts with cellular receptors, for example type S lectins, and generally any cellular receptor known to the human [The lectins, Properties, Functions and Applications in Biology and Medicine, IE Liener et al, Academy Press, N Y. 1986; Irwin J Goldstein et al., Adv. in Carbohydr. Chem. Biochem., 35 (1978, pp. 127-340). Examples that may be mentioned include the following sugars: pyranoses and furanoses, for example glucose, mannose, rhamnose, galactose or fructose, or maltose, lactose, sucrose, sucrose, fucose, cellobiose, allose, laminarabiose, gentiobiose, sophorose, melibiose, etc. Preferably, the sugar or sugars are chosen from glucose, mannose, rhamnose, galactose, fructose, lactose, sucrose and cellobiose, and can also be so-called "complex" sugars, that is to say several sugars coupled covalently with to each other, each sugar being preferably selected from the list cited above.

 For the purposes of the invention, the term "oligonucleotide" means chains containing one or more nucleotides, deoxynucleotides, ribonucleotides and / or deoxyribonucleotides which are monomeric units that differ from one another by the presence of bases which may be chosen from adenine, guanine,

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 cytosine, thymidine or uracil [see Lehninger Biochimie, Flammarion Medecine Sciences, 2nd Edition, p. 305-329].

Because of their property to form base pairs, oligonucleotides are widely used in molecular biology, for example as linkers, as probes, or to form constructs with DNA.

Moreover, the oligonucleotides can also be used in the form of conjugates, that is to say coupled to one or more other molecules having distinct properties.

By way of example, mention may be made of the coupling of an oligonucleotide with a reactive chemical group, with fluorescent or chemiluminescent groups, or with groups capable of promoting intermolecular interactions so as to promote entry into the cells. Such conjugates, described in Bioconjugate Chemistry [John Goodchild, Conjugates of Oligonucleotides and Modified Oligonucleotides: a Review of their Synthesis and Properties, Vol. 1, No. 3, 1990, pp.

165-187], have many uses and advantages such as, for example, the ability to improve the entry of complexes into the cells, to reduce the rate of degradation by nucleases, to increase the stability of the complex concerned, to follow the to become oligonucleotides in an organism etc ...

The oligonucleotides can be obtained according to standard methods known to those skilled in the art, and it is also possible to synthesize modified oligonucleotides according to the methods described in Bioconjugate Chemistry, John Goodchild, Conjugales of Oligonucleotides and Modified Oligonucleotides: a Review of their Synthesis and properties, Vol. 1, No. 3, 1990, pp. 165-187 or in Tetrahedron, Beaucage et al., The Synthesis of Modified Oligonucleotides by the Phosphoramiditeb Approach and Their Application, Vol. 49, No. 28, pp. 6123 to 6194.1993.

 Within the meaning of the invention, the term "peptide" means chains containing one or more amino acids linked together by peptide-type bonds [Lehninger Biochimie, Flammarion Medecine Sciences, 2nd Edition]. It can be the 20 "conventional" amino acids, that is to say those commonly found in the composition of proteins (Alamine, Valine, Leucine, Isoleucine, Proline, Phenylalamine, Tryptophan,

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 Methionine, Aspartic Acid, Glutamine, Lysine, Arginine, Histidine, Glycine, Serine, Threonine, Cysteine, Tyrosine, Asparagine, Glutamic Acid), or it may also be the so-called "rare" amino acids such as 4- hydroxyproline, desmosine, 5-hydroxylysine, N-methyllysine, 3-methylhistidine, isodesmosine, etc. Finally, it can also be amino acids appearing in different cells or various tissues in free or combined form and which are generally derived from α-amino acids (for example (3-alamine, y-aminobutyric acid, homocysteine, ornithine, canavanine, djenkalic acid, (3-cyanoalamin etc. .).

The peptides according to the invention may also be substituted at one or more of their functional groups, at the level of the carboxyl at a, of the amine function at a and / or at the level of the functional groups of the side chain of each of the amino acids. By way of example, mention may be made of substitutions with saturated or unsaturated, linear, branched or cyclic aliphatic groups containing 1 to 24 carbon atoms, such as, for example, cholesteryl, arachidonyl or retinoyl radicals, or monounsaturated groups. or polyaromatic such as, for example, benzyloxycarbonyl, benzyl ester or substituted or unsubstituted rhodaminyl derivatives. The interest of such substitutions falls within the framework of modifying the chemical and possibly biological properties of said peptides, for example in order to label them.

 Within the meaning of the invention, the term "marker" means any agent allowing identification, for example by analysis techniques such as fluorescence spectrometry, infrared spectrometry, nuclear magnetic resonance (NMR), etc., of This marker may also be a linear or cyclic peptide or pseudopeptide sequence comprising the Arg-Gly-Asp (Arginine-Glycine-Aspartic Acid) epitope for recognition of the primary and / or secondary receptors of the biotin, rhodamine and folate type. adhesion proteins of the integrin type.

 For the purposes of the invention, the polycation R is a linear or branched hydrophilic polycationic molecule capable of associating with the nucleic acid, which is polyanionic. For the purposes of the invention, it is understood to mean by association with the nucleic acid,

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dans laquelle : - Ri, R2 et R3 représentent indépendamment les uns des autres un atome d'hydrogène ou un groupement (CH2) qNR'R" avec q un nombre entier pouvant varier entre 1, 2,3, 4, 5 et 6 inclus, ceci de manière indépendante entre les différents groupements Ri, R2 et R3, - R' et R" représentent indépendamment l'un de l'autre un atome d'hydrogène ou un groupement (CH2)qNH2 avec q défini comme précédemment, et - m, n et p représentent indépendamment les uns des autres un nombre entier pouvant varier entre 0,1, 2,3, 4,5 et 6 inclus, avec lorsque n est supérieur à 1, m pouvant prendre des valeurs différentes et R3 des significations différentes au sein de la formule générale (II). any type of bonds, for example covalent bonds, electrostatic or ionic interactions, hydrogen bridges, etc. For example, the polycation R may be a polycationic molecule as defined in patent applications WO 96/17823, WO 97 / 18185, WO 97/31935, PCT / FR98 / 01041, and more generally throughout the literature concerning cationic lipid structures known to those skilled in the art. According to a preferred aspect of the invention, R represents a polyamine of general formula (II):

in which: R 1, R 2 and R 3 represent, independently of one another, a hydrogen atom or a group (CH 2) qNR 'R "with q an integer ranging between 1, 2,3, 4, 5 and 6 inclusive; in an independent manner between the different groups R 1, R 2 and R 3, R 'and R "represent, independently of one another, a hydrogen atom or a group (CH 2) qNH 2 with q defined as above, and m, n and p independently of one another represent an integer ranging from 0.1, 2.3, 4.5 and 6 inclusive, with when n is greater than 1, m being able to take different values and R3 having meanings different in the general formula (II).

Preferentially, R represents a spermine or a spermidine.

 An advantageous variant of the invention comprises the transfer agents of the invention are defined by the general formula (I) for which Y represents a hydrogen atom, and X represents a sugar as defined above.

 Another even more advantageous variant of the invention comprises the transfer agents of general formula (I) for which Y represents a hydrogen atom,

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 X represents a sugar, and R represents a polycation of general formula (II) as defined above.

 The terms x and y are defined in the general formula (I) so as to take any value between 10 and 22 inclusive. Preferably, x and y, independently of each other, are between 12 and 18 inclusive. More preferably, x and y are, independently of one another, 14, 15, 16, 17 or 18.

It is understood that the present invention also relates to the isomers of the products of general formula (I) when they exist, as well as their mixtures, or their salts
In particular, the compounds of the invention may be in the form of non-toxic and pharmaceutically acceptable salts. These non-toxic salts include salts with mineral acids (eg hydrochloric, sulfuric, hydrobromic, phosphoric, nitric acid), with organic acids (acetic acid, propionic acid, succinic acid, maleic acid, hydroxymaleic acid, benzoic acid, fumaric acid, methanesulfonic acid or oxalic), with mineral bases (sodium hydroxide, potassium hydroxide, lithium hydroxide, lime), or with organic bases (tertiary amines such as triethylamine, piperidine, benzylamine).

According to the invention, the products of general formula (I) are prepared by carrying out the following steps:
1) A first alkyl chain is prepared with x carbon atoms (x being defined as previously in the general formula (I)), comprising a hydroxy function and an ester function, by opening a corresponding lactone. The reaction is generally carried out in an alcohol, at basic pH and at a temperature of between -10 ° C. and room temperature. For example, the alcohol may be methanol or ethanol.

 2) Then, the sugar is condensed on the bifunctional alkyl chain obtained in the previous step. The condensation is advantageously carried out in a solvent

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 chlorine, such as, for example, dichloromethane or chloroform, and in the presence of a Lewis acid, at a temperature of between -5 ° C. and 10 ° C. The lewis acid may, for example, be chosen from tin chloride, iron chloride, p-toluenesulphonic acid (tsOH), trimethylsilyltrifluoromethanesulfonic acid (TMStf), boron trifluoride etherate, etc. [Kazunobu Toshima et al., Recent Progress in O-glcosilation Methods and their Application to Natural Products Synthesis, Chem. Rev. 1993, Vol.

93, pp. 1503-1531].

 3) In a third step, the ester function present on the bifunctional chain is hydrolysed in an acidic function according to the known methods.

In particular, the procedure is carried out in a basic medium in a high-boiling alcohol at a temperature of between 50 ° C. and the reflux temperature of the reaction mixture.

4) Then, an alkylamine chain of general formula (III):
H2N- (CH2) yY (III) in which y and Y are defined as above in the general formula (I), is coupled to the compound obtained in the preceding step, according to conventional peptide coupling methods (Bodanski M., Principles and Practices of Peptides Synthesis, Ed. Springe-Verlag) or any similar method known to those skilled in the art.

In particular, the reaction is generally carried out in the presence of a nonnucleophilic base in suitable aprotic solvents, at a temperature of between 0 and 100 ° C., the pH being adjusted between 9 and 11.

By way of example, chloroform, dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane, toluene or benzene may be used as the solvent.

The non-nucleophilic bases employed are preferably tertiary amines, calcium carbonate or sodium dicarbonate. Even more preferentially, the bases used are tertiary amines, for example triethylamine (TEA) or N-ethyldiisopropylamine.

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 Advantageously, the peptide coupling is carried out between 0 and 50 ° C., and preferably between 10 and 30 ° C.

 5) The amide obtained in the previous step is then reduced to amine. This is done according to conventional methods known to those skilled in the art. For example, one operates in an anhydrous organic solvent such as anhydrous tetrahydrofuran, by action of lithium aluminum hydride LiAlH4. Other reducing agents that may be used are, for example, borane, borane in dimethylsulfide (BH3-SMe2), sodium borohydride / titanium tetrachloride (NaBH4, TiCl4), phosphorus oxide chloride on zinc (POCl3 / Zn), phosphorus pentasulfide (P4S10) on Raney nickel, etc. [Richard C. Larock, Comprehensive Organic Transformations, VCH Publishers Inc., 1989]. It can also be operated by catalytic hydrogenation.

Advantageously, the reduction is carried out by action of lithium aluminum hydride LiAlH 4 in anhydrous tetrahydrofuran at the reflux temperature of the mixture.

pour laquelle X, Y, x et y sont définis comme précédemment. A compound of general formula (IV) is thus obtained:

for which X, Y, x and y are defined as before.

 6) Finally, in a last step, the acid derivative corresponding to the polycation R as defined above, is coupled to the compound of general formula (IV) obtained in the preceding step, according to conventional peptide coupling methods (Bodanski M., Principles and Practices of Peptides Synthesis, Ed. Springe-Verlag) or any similar method known to those skilled in the art.

In particular, the reaction is generally carried out in the presence of a nonnucleophilic base in suitable aprotic solvents, at a temperature of between 0 and 100 ° C., the pH being adjusted between 9 and 11.

By way of example, chloroform, dimethylformamide, methylpyrrolidone, acetonitrile, dichloromethane, toluene or benzene may be used as the solvent.

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The non-nucleophilic bases employed are preferably tertiary amines, calcium carbonate or sodium dicarbonate. Even more preferentially, the bases used are tertiary amines, for example triethylamine (TEA) or N-ethyldiisopropylamine.

Advantageously, the peptide coupling is carried out between 0 and 50 ° C., and preferably between 10 and 30 ° C.

The acid derivatives corresponding to the polycation are commercially available.

 Of course, when the substituents of sugar and / or polycation can interfere with the reaction, it is preferable to protect them beforehand with compatible radicals that can be put in place and removed without affecting the rest of the molecule. This is done according to the conventional methods known to those skilled in the art, and in particular according to the methods described in W. GREENE, Protective Groups in Organic Synthesis, 2nd Edition, Wiley-Interscience, in McOMIE, Protective Groups in Organic Chemistry, Plenum Press (1973), or in Philip J Kocienski, Protecting Groups, Thieme.

 Furthermore, each step of the preparation process can be followed, if necessary, steps of separation and purification of the compound obtained according to methods known to those skilled in the art.

By way of illustrative example of advantageous nucleic acid transfer agents according to the invention, mention may be made of the following compounds:
Compound 1

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Another subject of the invention relates to compositions comprising a nucleic acid transfer agent as defined above, and a nucleic acid. The respective amounts of each component can be adjusted easily by those skilled in the art depending on the transfer agent used, the nucleic acid, and the desired applications (including the type of cells to be transfected).

 Within the meaning of the invention, the term "nucleic acid" is intended to mean both a deoxyribonucleic acid and a ribonucleic acid. It may be natural or artificial sequences, and in particular genomic DNA (gDNA) or complementary DNA (cDNA). ), messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA (rRNA), hybrid sequences or synthetic or semi-synthetic sequences, modified or unmodified oligonucleotides. These nucleic acids may be of human, animal, plant, bacterial or viral origin, etc. They may be obtained by any technique known to those skilled in the art, and in particular by screening of libraries, by chemical synthesis, or else by mixed methods including the chemical or enzymatic modification of sequences obtained by screening libraries. They can be chemically modified.

 With particular reference to deoxyribonucleic acids, they can be single or double-stranded as well as short oligonucleotides or longer sequences. In particular, the nucleic acids are advantageously constituted by plasmids, vectors, episomes, expression cassettes, etc. These deoxyribonucleic acids may carry an origin of replication functional or otherwise in the target cell, one or more marker genes, regulatory sequences of transcription or replication, genes of therapeutic interest, sequences

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 modified or unmodified antisense, binding regions to other cellular components, etc.

 Preferably, the nucleic acid comprises one or more genes of therapeutic interest under the control of regulatory sequences, for example one or more promoters and a transcriptional terminator active in the target cells.

 For the purposes of the invention, the term "gene of therapeutic interest" is intended to mean any gene coding for a protein product having a therapeutic effect. The protein product thus coded may in particular be a protein or a peptide. This protein product may be exogenous homologous or endogenous with respect to the target cell, that is to say a product that is normally expressed in the target cell when it has no pathology. In this case, the expression of a protein makes it possible for example to overcome insufficient expression in the cell or the expression of an inactive or weakly active protein due to a modification, or to overexpress said protein. therapeutic interest can also encode a mutant of a cellular protein, having increased stability, modified activity, etc.

The protein product may also be heterologous with respect to the target cell. In this case, an expressed protein may for example supplement or provide a deficient activity in the cell, enabling it to fight against a pathology, or to stimulate an immune response.

 Among the therapeutic products within the meaning of the present invention, mention may be made more particularly of enzymes, blood derivatives, hormones, lymphokines: interleukins, interferons, TNF, etc. (FR 92/03120), growth factors , neurotransmitters or their precursors or synthetic enzymes, trophic factors (BDNF, CNTF, NGF, IGF, GMF, aFGF, bFGF, NT3, NT5, HARP / pleiotrophin, etc.) apolipoproteins (ApoAI, ApoAIV, ApoE , etc ..., FR 93/05125), dystrophin or a minidystrophin (FR 91/11947), CFTR protein associated with cystic fibrosis, tumor suppressor genes (p53, Rb, RaplA, DCC, k-rev, etc ..., FR 93/04745), the genes encoding factors involved in the

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 coagulation (Factors VII, VIII, IX), genes involved in DNA repair, suicide genes (thymidine kinase, cytosine deaminase), hemoglobin genes or other protein transporters, metabolic enzymes, catabolism etc ...

 The nucleic acid of therapeutic interest may also be an antisense gene or sequence whose expression in the target cell makes it possible to control the expression of genes or the transcription of cellular mRNAs. Such sequences may, for example, be transcribed into the target cell into RNAs complementary to cellular mRNAs and thus block their translation into protein, according to the technique described in patent EP 140 308. Therapeutic genes also include sequences coding for ribozymes, which are capable of selectively killing target RNAs (EP 321 201).

 As indicated above, the nucleic acid may also comprise one or more genes encoding an antigenic peptide capable of generating an immune response in humans or animals. In this particular embodiment, the invention allows the production of vaccines or immunotherapeutic treatments applied to humans or animals, especially against microorganisms, viruses or cancers. It may be in particular antigenic peptides specific for Epstein Barr virus, HIV virus, hepatitis B virus (EP 185 573), pseudo-rabies virus, syncitia-forming virus, other viruses or tumor-specific antigenic peptides (EP 259,212).

 Preferably, the nucleic acid also comprises sequences allowing the expression of the gene of therapeutic interest and / or the gene coding for the antigenic peptide in the cell or the desired organ. These may be sequences that are naturally responsible for the expression of the gene when these sequences are likely to function in the infected cell. It can also be sequences of different origin (responsible for the expression of other proteins, or even synthetic). In particular, they may be gene promoter sequences

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 For example, eukaryotic or viral proteins may be promoter sequences derived from the genome of the cell that it is desired to infect. Similarly, they may be promoter sequences derived from the genome of a virus. In this respect, mention may be made, for example, of the promoters of the E1A, MLP, CMV, RSV genes, etc. Moreover, these expression sequences may be modified by addition of activation, regulation sequences, etc. It can also be promoter, inducible or repressible.

 Moreover, the nucleic acid may also comprise, in particular upstream of the gene of therapeutic interest, a signal sequence directing the therapeutic product synthesized in the secretory pathways of the target cell. This signal sequence may be the natural signal sequence of the product. therapeutic, but it can also be any other functional signal sequence, or an artificial signal sequence The nucleic acid may also include a signal sequence directing the synthesized therapeutic product to a particular compartment of the cell.

 The compositions according to the invention may further comprise one or more adjuvants capable of associating with the transfer agent / nucleic acid complexes and of improving their transfecting capacity. In another embodiment, the present invention therefore relates to compositions comprising a nucleic acid, a nucleic acid transfer agent as defined above and at least one adjuvant capable of associating with the transfer agent complexes. nucleic acid and to improve its transfecting power. The presence of this type of adjuvant (lipids, peptides or proteins, for example) may advantageously make it possible to increase the transfecting power of the compounds. In this context, the compositions of the invention may comprise, as an adjuvant, one or more neutral lipids.

 More preferably, the neutral lipids used in the context of the present invention are lipids with two fatty chains. Particularly advantageously, natural or synthetic lipids, zwitterionic or devoid of ionic charge are used under physiological conditions. It may be chosen more particularly from dioleoylphosphatidylethanolamine (DOPE),

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 oleoylpalmitoylphosphatidylethanolamine (POPE), distearoyl, -palmitoyl, -mirystoylphosphatidylethanolamines and their N-methylated derivatives 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as especially sphingomyelins) or asialogangliosides (such as in particular asialoGM1 and GM2).

 These different lipids can be obtained either by synthesis, or by extraction from organs (example: the brain) or eggs, by conventional techniques well known to those skilled in the art. In particular, the extraction of natural lipids can be carried out using organic solvents (see also Lehninger, Biochemistry).

 More recently, the Applicant has demonstrated that it is also particularly advantageous to use as an adjuvant, a compound that may or may not be directly involved in the condensation of said nucleic acid, such as those described in the patent application WO 96 / 25508. The presence of such a compound, within a composition according to the invention, makes it possible to reduce the amount of transfecting agent, with the beneficial consequences which result from it toxicologically, without causing any damage to the activity. transfecting. By compound involved in the condensation of the nucleic acid is meant to define a compacting compound, directly or otherwise, the nucleic acid. More precisely, this compound can either act directly at the level of the nucleic acid to be transfected or can intervene at the level of an auxiliary compound which is directly involved in the condensation of this nucleic acid. Preferably, it acts directly at the level of the nucleic acid. In particular, the precompacting agent may be any polycation, for example polylysine. According to a preferred embodiment, the agent involved in the condensation of the nucleic acid is derived in whole or in part from a protamine, a histone, or a nucleolin and / or from one of their derivatives. Such an agent may also consist, in whole or in part, of peptide motifs (KTPKKAKKP) and / or (ATPAKKAA), the number of the motifs being

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 vary between 2 and 10. In the structure of the compound according to the invention, these patterns can be repeated continuously or not. Thus they can be separated by links of a biochemical nature, for example by one or more amino acids, or of a chemical nature.

 Preferably, the compositions of the invention comprise from 0.01 to 20 equivalents of adjuvant for one nucleic acid equivalent in mol / mol and, more preferably, from 0.5 to 5.

 In a particularly advantageous embodiment, the compositions according to the present invention further comprise a targeting element for orienting the transfer of the nucleic acid. This targeting element may be an extracellular targeting element to guide the transfer of DNA to certain desired cell types or tissues (tumor cells, liver cells, hematopoietic cells, etc.). It can also be an intracellular targeting element to guide the transfer of nucleic acid to certain preferred cellular compartments (mitochondria, nucleus, etc.). The targeting element may be linked to the nucleic acid transfer agent according to the invention, or also to the nucleic acid as has been specified above. When the targeting element is linked to the nucleic acid transfer agent of general formula (I), it is preferably incorporated at the level of the substituent X or Y.

 Among the targeting elements that may be used in the context of the invention, mention may be made of sugars, peptides, proteins, oligonucleotides, lipids, neuromediators, hormones, vitamins or their derivatives. Preferentially, these are sugars, peptides or proteins such as antibodies or antibody fragments, cell receptor ligands or fragments thereof, receptors or receptor fragments, etc. In particular, they may be growth factor receptor ligands, cytokine receptors, cell lectin type receptors, or RGD sequence ligands with affinity for adhesion protein receptors such as integrins. can also quote the

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 receptors for transferrin, HDL and LDL, or the folate transporter.

The targeting element may also be a sugar that makes it possible to target lectins such as asialoglycoprotein or syalide receptors such as sialyl Lewis X, or an antibody Fab fragment, or a single chain antibody (ScFv).

 The combination of the targeting elements with nucleolipid complexes can be carried out by any technique known to those skilled in the art, for example by coupling to a hydrophobic part or to a part which interacts with the nucleic acid of the transfer agent according to the invention, or to a group which interacts with the transfer agent according to the invention or with the nucleic acid. The interactions in question may be, according to a preferred mode, of ionic or covalent nature.

 The subject of the invention is also the use of the compounds as defined above for the transfer of polynucleotides (and more generally polyanions) into cells in vitro, in vivo or ex vivo. More specifically, the subject of the present invention is the use of the compounds as defined above for the preparation of a medicament intended to treat diseases resulting from a deficiency in a protein or nucleic product. The polynucleotide contained in said drug encodes said protein or nucleic product, or constitutes said nucleic product, capable of correcting said diseases in vivo or ex vivo.

 For uses in vivo, for example in therapy or for the study of the regulation of genes or the creation of animal models of pathologies, the compositions according to the invention can be formulated for topical, cutaneous, oral administrations rectal, vaginal, parenteral, intranasal, intravenous, intramuscular, subcutaneous, intraocular, transdermal, intratracheal, intraperitoneal, etc. Preferably, the compositions of the invention contain a pharmaceutically acceptable carrier for an injectable formulation, especially for direct injection at the desired organ, or for topical administration (on skin and / or mucosa). It may be in particular sterile solutions, isotonic, or dry compositions, especially freeze-dried, which, by

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 addition according to the case of sterilized water or physiological saline, allow the constitution of injectable solutions. The doses of nucleic acids used for the injection as well as the number of administrations can be adapted according to various parameters, and in particular according to the mode of administration used, the pathology concerned, the gene to be expressed, or the duration of the desired treatment. As regards more particularly the mode of administration, it can be either a direct injection into the tissues, for example at the level of the tumors, or in the circulatory ways, or of a cell treatment in culture followed their reimplantation in vivo, by injection or graft. The tissues concerned in the context of the present invention are for example the muscles, the skin, the brain, the lungs, the liver, the spleen, the bone marrow, the thymus, the heart, the lymph, the blood, the bones, cartilages, pancreas, kidneys, bladder, stomach, intestines, testes, ovaries, rectum, nervous system, eyes, glands, connective tissue, etc.

The invention further relates to a method of transferring nucleic acids into cells comprising the steps of: (1) contacting the nucleic acid with a transfer agent as defined above to form a complex, and (2) contacting the cells with the complex formed in (1)
The cells can be brought into contact with the complex by incubating the cells with said complex (for in vitro or ex vivo uses), or by injecting the complex into an organism (for in vivo uses). The incubation is preferably carried out in the presence of, for example, from 0.01 to 1000 g of nucleic acid per 10 6 cells. For in vivo administration, doses of nucleic acid ranging from 0.01 to 10 mg may for example be used
In the case where the compositions of the invention additionally contain one or more adjuvants as defined above, the adjuvant (s) are premixed with the transfer agent according to the invention and / or with the nucleic acid.

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 The present invention thus provides a particularly advantageous method for the transfer of nucleic acids in vivo, in particular for the treatment of diseases, comprising the in vivo or in vitro administration of a nucleic acid coding for a protein or capable of being transcribed into a protein. nucleic acid capable of correcting said disease, said nucleic acid being associated with a compound of general formula (I) under the conditions defined above.

 The nucleic acid transfer agents of the invention are particularly useful for the transfer of nucleic acids into primary cells or into established lines. It can be fibroblastic, muscle, nerve cells (neurons, astrocytes, glial cells), liver cells, hematopoietic cells (lymphocytes, CD34, dendritic cells, etc.), epithelial cells, etc. , in differentiated or pluripotent form (precursors).

 In addition to the foregoing, the present invention also includes other features and advantages which will emerge from the examples and figures which follow, and which should be considered as illustrating the invention without limiting the scope thereof. In particular, the Applicant proposes, without limitation, various operating protocols as well as reaction intermediates that can be used to prepare the transfer agents of general formula (I). Of course, it is within the abilities of those skilled in the art to draw inspiration from these protocols or intermediate products to develop analogous processes to lead to these same compounds. It is also a matter for the person skilled in the art to draw inspiration from the synthetic processes described in the various patent applications mentioned above for the synthesis of the polycation R included in the general formula (I) (WO 96/17823, WO 97/18185). , WO 97/31935 ...).

FIGURES Figure 1. Schematic representation of plasmid pXL2774 used in DNA transfer experiments in cells.

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 FIG. 2: In vitro gene transfer activity in HeLa cells of complexes formed from compound 2 according to the invention without co-lipid, or in the presence of cholesterol and in the presence of DOPE as co-lipids. ordinates represents the expression of luciferase in pg / well. The axis of absisses indicates the lipid / DNA ratio in nmol / g of DNA.

Figure 3: In vivo gene transfer activity after direct injection into the anterior muscle of mouse tibias of complexes formed from compound 2 according to the present invention in the presence of DOPE (1 1). The ordinate axis indicates the expression of luciferase in pg / muscle. The axis of absisses indicates the compound ratio 2 / DNA in nmoles / g of DNA.

EXAMPLES Materials and Methods a) Material # The starting polyamines, such as spermidine, spermine, tris- (2-aminoethyl) amine, phenylenediamine, diaminoalkane, etc., are commercially available or they can be synthesized by conventional methods (for example by cyanoethylation of amines commercially available to obtain branched amines) # of numerous compounds such as, for example, triethylamine, benzotriazol-1-yloxytris (dimethylamino) phosphonium hexafluorophosphate (BOP), chloroformate benzyl, 11-bromoundecanol, etc. are also commercial products.

 # Amberlite IR 120 is a commercial ion exchange resin (BDH catalog).

 Dimethylsulfoxide (DMSO), previously treated with potassium hydroxide, was distilled over calcium hydride and then stored on a 4Å molecular sieve.

 Dichloromethane was distilled over phosphorus pentoxide and stored on a 4Å molecular sieve.

 # Tetrahydrofuran (THF) was distilled over sodium in the presence of benzophenone.

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 * For reactions requiring anhydrous conditions, all the glassware is flame dried under a stream of nitrogen. b) Methods - Spectroscopic Analyzes The nuclear magnetic resonance spectra (NMR) were recorded on a Brucker MSL 30 spectrometer at the frequency of 300 MHz for the proton and 75 MHz for the carbon. All chemical shifts are reported in ppm relative to the frequency of tetramethylsilane (TMS). The spectra were recorded using either the TMS or the residual solvent signal as internal reference. The multiplicity of signals is denoted by the following abbreviations: s (singlet), d (doublet), t (triplet), q (quadruplet), and m (multiplet).

- Chromatography techniques # The kinetics of the reactions were followed by thin layer chromatography (TLC) with a silica gel containing a fluorescent indicator (Merck Slilicagel 60
F254) as support. The chromatograms were revealed by spraying with an alcoholic solution of anisaldehyde.

 # All the column chromatographies were carried out under pressure of compressed air with silica gel 60 as a stationary phase (0.05-0.02 mm). The mobile phase employed differs according to the type of synthesis (medium pressure chromatography).

# HPLC (High Performance Liquid Chromatography) analyzes are carried out on a Waters LC 4000 device equipped with a C4 type analytical column marketed by Applied Biosystem ("Brownlee Columns" in stainless steel).
3 cm in length and 0.46 cm in diameter) and a "Waters 486" detector at 220 nm.
The stationary phase is butyl aquapore 7 microns, and the mobile phases are deionized water (2500 cm3) or acetonitrile (2500 cm3) supplemented with trifluoroacetic acid (2.5 cm3). The flow rate is 1 ml per minute

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méthylène-carbamoyl}-15-pentadécanyl-16-octadécyIJ--P-D-mannopyrannose (Composé 1) a) Synthèse du 1-ol pentadécanoate de méthyle A 10 g de pentadécalactone (41,60 mmol) dans 41,60 cm3 de méthanol, on ajoute 6,66 cm3 de méthylate de sodium 2N (13,31 mmol) à 0 C. EXAMPLE OF TRANSFECTION AGENTS Example 1 synthesis of 1 '- [- (3- [4- (3-aminopropylamino) butylamino] -

methylene-carbamoyl} -15-pentadecanyl-16-octadecyl-PD-mannopyranose (Compound 1) a) Synthesis of methyl 1-ol pentadecanoate to 10 g of pentadecalactone (41.60 mmol) in 41.60 cm3 of methanol, 6.63 cm3 of 2N sodium methoxide (13.31 mmol) is added at 0 ° C.

After 9 hours, 9.24 cm3 of acetic acid are added and allowed to act for 15 minutes. The solution is then evaporated to dryness under vacuum, the residue is then taken up in dichloromethane and the sodium bicarbonate is washed. The organic phase obtained is dried with magnesium sulphate and the solvent is evaporated in a rotary evaporator. The purification is carried out in a hexane / ethyl acetate mixture 6: 4. obtains 1-ol-pentadecanoate methyl with a yield of 80%.

b) Synthèse du r-rpentadécan-15-oate de méthyn-2\3\4\6'-tétra-0-acétyt-6-D- mannopyrannose A O C, on ajoute 5,26 cm3 de chlorure d'étain (44,94 mmol) à 8,72 g de mannose peracétylé (22,47 mmol) dans 56 cm3 de dichlorométhane pendant 30 minutes. Puis on ajoute 7,34 g de 1-ol pentadécanoate de méthyle précédemment obtenu en a) (26,96 mmol). Après 2 heures, le mélange réactionnel est dilué par de l'éther éthylique et on verse dans une solution d'hydrogénophosphate de sodium (NaHP04). Les phases aqueuses sont extraites avec du diéthyléther et les phases organiques sont successivement lavées par une solution de carbonate de sodium, de la saumure, puis séchées sur sulfate de magnésium. Le produit obtenu après évaporation sous vide à sec est purifié par chromatographie moyenne pression dans un mélange heptane/acétate d'éthyle 7 :3. Le rendement est de 53 %. 1H NMR (CDCl3): # (ppm) 1.26 (m, 12H, (CH2) 10), 1.5-1.6 (m, 4H, H-2 and H-13), 2.30 (t , 2H, J = 7. 60 Hz, H-14), 3.64 (t, 1H, J = 5.84 Hz, H1), 3.67 (s, 3H, H-16).

b) Synthesis of methyn-2β, 6'-tetra-O-acetyl-6-D-mannopyranose r-pentadecan-15-oate was added 5.26 cm 3 of tin chloride (44, 94 mmol) to 8.72 g of peracetyl mannose (22.47 mmol) in 56 cm3 of dichloromethane for 30 minutes. 7.34 g of methyl 1-ol pentadecanoate previously obtained in (a) (26.96 mmol) are then added. After 2 hours, the reaction mixture is diluted with ethyl ether and poured into a solution of sodium hydrogenphosphate (NaHPO 4). The aqueous phases are extracted with diethyl ether and the organic phases are successively washed with sodium carbonate solution, brine and then dried over magnesium sulfate. The product obtained after evaporation in a dry vacuum is purified by medium pressure chromatography in a heptane / ethyl acetate mixture 7: 3. The yield is 53%.

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1H NMR (CDCl3): # (ppm) 1.26 (m, 12H, (CH2) 10), 1.59 (m, 4H, H-2 and H-13), 2.01, 2.05, 2 , 12 and 2.17 (s, 3H, OCOCH 3), 2.29 (t, 2H, J = 7.62 Hz, H-14), 3.40 (m, 1H, J = 7.89 Hz, H -la), 3.66 (m, 1H, J = 7.83Hz, H-1b), 3.67 (s, 3H, H-16), 3.97-4.05 (m, 2H, H). -4 'and H-5'), 4.1 (dd, 1H, J = 5.57 Hz and 12.32 Hz, H-6'a), 4.29 (dd, 1H, J = 5.57 Hz and 12.32 Hz, H-6'b), 4.8 (d, 1H, J = 1.85 Hz, H-1 '), 5.23 (dd, 1H, J = 1.85 Hz and 3.23 Hz, H-2 '), 5.35 (dd, 1H, J = 9.97 Hz and 3.23 Hz, H-3') c) Synthesis of methyl r-fpentadecan-15-oate] -ss-D-mannopyranose 3.63 g of product obtained in the previous step (6.01 mmol) in solution in 12 cm3 of methanol are treated with 3 cm3 of 2N sodium methoxide (6.01 mmol). When the reaction is complete, the latter is neutralized with Amberlite IR120 (1 equivalent weight / volume), filtered and evaporated to dryness under vacuum.

d) Synthèse du l'-[pentadécan-15-oate de méthyll-2',3',4',6'-tétra-O-benzyl-(3- D-mannopyrannose A 2 g (4,56 mmol) de produit obtenu à l'étape précédente c) en solution dans 20 cm3 de diméthylformamide (DMF) anhydre, on ajoute successivement 4,54 g d'iodure de potassium (27,36 mmol), 1,09 g d'hydrure de sodium 60% (27,36 mmol) et 3,25 cm3 de bromure de benzyle (27,36 mmol). Après 12 heures, on ajoute 18,24 cm3 d'une solution saturée de chlorure d'ammonium, et on laisse agir pendant 10 minutes Puis, on dilue avec de l'eau et on extrait la phase organique par de l'acétate d'éthyle Celleci est ensuite lavée avec de l'eau et de la saumure, et est finalement séchée par du sulfate de magnésium. On effectue par ailleurs un lavage supplémentaire par une solution saturée de thiosulfate de sodium afin d'éliminer les ions iodures. On évapore sous vide et on purifie l'huile résultante dans un mélange heptane/acétate d'éthyle 9:1 Le produit est obtenu avec un rendement de 60 % 1H NMR (CDCl3): # (ppm) 1.28 (m, 12H, (CH2) 10), 1.59 (m, 4H, H-2 and H-13), 2.34 (t, 2H, J). = 7.62 Hz, H-14), 3.41 (m, 1H, J = 6.71 Hz, H-1a), 3.74 (m, 1H, J = 6.71 Hz, H-1 b) , 3.67 (s, 3H, H-16), 3.5-3.82 (m, 6H, H-2 ', H-3', H-4 ', H-5' and H-6 ' ), 4.75 (d, 1H, J = 1.72 Hz, H-1 ').

d) Synthesis of Methyl-2 ', 3', 4 ', 6'-tetra-O-benzyl- (3-D-mannopyranose) - [pentadecan-15-oate A 2 g (4.56 mmol) product obtained in the preceding step c) in solution in 20 cm3 of anhydrous dimethylformamide (DMF), 4.54 g of potassium iodide (27.36 mmol), 1.09 g of sodium hydride, are added successively. % (27.36 mmol) and 3.25 cc of benzyl bromide (27.36 mmol). After 12 hours, 18.24 cm3 of a saturated solution of ammonium chloride are added and the mixture is allowed to act for 10 minutes. Then, it is diluted with water and the organic phase is extracted with sodium acetate. This is then washed with water and brine, and finally dried with magnesium sulfate. In addition, an additional washing is carried out with a saturated solution of sodium thiosulfate in order to eliminate the iodide ions. It is evaporated under vacuum and the resulting oil is purified in a 9: 1 heptane / ethyl acetate mixture. The product is obtained in a yield of 60%.

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 1H NMR (CDCl3): d (ppm) 1.28 (m, 14H, (CH2) 10), 1.49 (m, 2H, H-2), 1.59 (m, 2H, H-13), 2.31 (t, 2H, J = 7.62 Hz, H-14), 3.34 (m, 1H, J = 6.71 Hz, H-la), 3.63 (m, 1H, J = 6.71 Hz, H-lb), 3.67 (s, 3H, H-16), 3.75 (m, 1H, J = 8.97 Hz and 6.21 Hz, H-5 '), 3 , 78 (s, 2H, CH 2 C 6 H 6), 3.90 (dd, 1H, J = 6.21 Hz and J = 11.82 Hz, H-6'a), 3.97 (dd, 1H, J = 6). , 21 Hz and J = 11.82 Hz, H-6'b), 4.07 (s, 2H, CH2C6H6), 4.52 (dd, J = 2.91 Hz and 7.83 Hz, H-3 4.57 (s, 2H, CH2C6H6), 4.63 (s, 2H, CH2C6H6), 4.69 (dd, 1H, J = 2.52 Hz and 2.91 Hz, H-2 '). , 4.74 (1H, J = 2.52 Hz, H-1 '), 4.85 (dd, 1H, J = 7.83 Hz and 8.97 Hz, H-4'),, 7.35 (m, 18H, C6H6). e) Synthesis of 1 '- [pentadecan-15-oic] -2', 3 ', 4', 6'-tetra-O-benzyl-ss-Dmannopyranose A 0.50 g (0.73 mmol) of the product obtained in the previous step d) in solution in 7 cm3 of methanol, 4.68 cm3 of a 25% sodium hydroxide solution are added. The reaction mixture is refluxed for 30 minutes. Then, the cold mixture is neutralized with a 5% hydrochloric acid solution. The organic phase is extracted with ethyl acetate and evaporated to dryness under vacuum. The purification is carried out in a heptane / ethyl acetate mixture 4: 6. product is obtained with a yield of 62%.

f) Synthèse du l'-(pentadécan-15-carbamoyl-16-octadécyll-2',3',4',6'-tétra-O- benzyl-ss-D-mannopyrannose A 0,29 g (0,37 mmol) d'une solution de produit obtenu à l'étape précédente e), en solution dans 5 cm3 de chloroforme, on ajoute successivement du 0,23 g de BOP 1H NMR (CDCl3): d (ppm) 1.28 (m, 14H, (CH2) 10), 1.49 (m, 2H, H-2), 1.59 (m, 2H, H-13), 2.34 (t, 2H, J = 7.62 Hz, H-14), 3.34 (m, 1H, J = 6.71 Hz, H-la), 3.63 (m, 1H, J = 6.71 Hz, H-lb), 3.75 (m, 1H, J = 8.97 Hz and 6.21 Hz, H-5 '), 3.78 (s, 2H, CH2C6H6), 3.90 (dd, 1H, J = 6.21 Hz and J = 11.82 Hz, H-6'a), 3.97 (dd, 1H, J = 6.21 Hz and J = 11, 82 Hz, H- 6'b), 4.07 (s, 2H, CH 2 C 6 H 6), 4.52 (dd, J = 2.91 Hz and 7.83 Hz, H-3 '), 4.57 (s, 2H, CH 2 C 6 H 6) , 4.63 (s, 2H, CH2C6H6), 4.69 (dd, 1H, J = 2.52 Hz and 2.91 Hz, H-2 '), 4.74 (1H, J = 2.52 Hz. , H-1 '), 4.85 (dd, 1H, J = 7.83 Hz and 8.97 Hz, H-4'),, 7.35 (m, 18H, C6H6).

f) Synthesis of - (pentadecan-15-carbamoyl-16-octadecyl-2 ', 3', 4 ', 6'-tetra-O-benzyl-ss-D-mannopyranose A 0.29 g (0.37 g) mmol) of a solution of product obtained in the previous step e), dissolved in 5 cm3 of chloroform, 0.23 g of BOP are added successively.

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 (0.52 mmol), 0.21 cm3 of diisopropylethylamine (1.48 mmol) and 0.12 g of octadecylamine (0.44 mmol). When the reaction is complete, it is diluted with dichloromethane and washed with water, then dried with magnesium sulfate and evaporated to dryness under vacuum. The product obtained is purified by medium pressure chromatography in a heptane mixture. / ethyl acetate 6: 4. product is obtained with a yield of 98%.

z) Synthèse du r-fpentadécan-15-amino-16-octadécYl1-2',3',4',6'-tétra-0- benzyl-ss-D-mannopyrannose A 0,77 g (0,75 mmol) de produit obtenu à l'étape précédente f), dans 15 cm3 de tétrahydrofuranne (THF) anhydre, on ajoute 0,056 g d'hydrure de lithium aluminium AlLiH4 (1,50 mmol). On chauffe à reflux pendant 10 heures Puis, le mélange réactionnel est refroidi dans un bain de glace et on ajoute 56 l d'eau, puis 112 fil de soude 2N après 10 minutes, et enfin encore 56 l d'eau 10 minutes plus tard. On filtre et on évapore à sec sous vide. Le produit obtenu est purifié dans un mélange de dichlorométhane/méthanol/ammoniac 28 % 9:2 0,5. Le produit est obtenu avec un rendement de 86 %. 1H NMR (CDCl3): δ (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.27 (m, 14H, (CH2) 25), 1.47 (m, 4H, H-2 and H-17), 1.58 (m, 2H, H-13), 2.13 (t, 2H, J = 7.92 Hz, H-14), 3.23 (m, 2H, H-16), 3.34 (m, 1H, J = 6.71Hz, H-1a), 3.63 (m, 1H, J = 6.71Hz, H-1b), 3.75 (m, 1H, J = 8.97 Hz and 6.21 Hz, H-5 '), 3.78 (s, 2H, CH 2 C 6 H 6), 3.90 (dd, 1H, J = 6.21 Hz and = 11.82 Hz, H-6'a), 3.97 (dd, 1H, J = 6.21 Hz and J = 11.82 Hz, H-6'b), 4.07 (s, 2H, CH2C6H6), 4.52 (dd, J = 2.91 Hz and 7.83 Hz, H-3 '), 4.57 (s, 2H, CH2C6H6), 4.63 (s, 2H, CH2C6H6), 4 , 69 (dd, 1H, J = 2.52 Hz and 2.91 Hz, H-2 '), 4.74 (1H, J = 2.52 Hz, H-1'), 4.85 (dd, 1H, J = 7.83 Hz and 8.97 Hz, H-4 '), 5.37 (band, 1H, HNCO), 7.35 (m, 18H, C6H6).

z) Synthesis of r-pentadecan-15-amino-16-octadecyl-2 ', 3', 4 ', 6'-tetra-O-benzyl-s-D-mannopyranose A 0.77 g (0.75 mmol) of product obtained in the preceding step f), in 15 cm3 of anhydrous tetrahydrofuran (THF), 0.056 g of lithium aluminum hydride AlLiH4 (1.50 mmol) is added. The mixture is heated under reflux for 10 hours. The reaction mixture is then cooled in an ice bath and 56 l of water are added, then 112 l of 2N sodium hydroxide after 10 minutes, and finally 56 l of water 10 minutes later. . Filtered and evaporated to dryness under vacuum. The product obtained is purified in a mixture of dichloromethane / methanol / ammonia 28% 9: 2 0.5. The product is obtained with a yield of 86%.

1H NMR (CDCl3): δ (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.27 (m, 14H, (CH2) 25), 1.4-1, 6 (m, 9H, H-2, H-17, H-14, H-17 and NH), 2.57 (t, 4H, J = 7.92 Hz, H-15 and H-16), 3, 34 (m, 1H, J = 6.71Hz, H-1a), 3.63 (m, 1H, J = 6.71Hz, H-1b), 3.75 (m, 1H, J = 8, 97 Hz and 6.21 Hz, H-5 '), 3.78 (s, 2H, CH2C6H6), 3.90 (dd, 1H, J = 6.21

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h) Synthèse du l'-f-(3-f4-(3-tert-butoxycarbonvl-amino-propyl-tertbutoxvcarbonvl-amino)-butyl-tert-butoxvcarbonyl-aminol-méthvlènecarbamovl)-15-pentadécanvl-16-octadécvll-2' ,3' ,4' ,6' -tétra-O-benzvl-B-D- mannopyrannose A 0,63 g (0,61 mmol) d'une solution de produit obtenu précédemment à létape g), dans 10 cm3 de chloroforme, on ajoute successivement 0,38 g de BOP (0,85 mmol), 0,425 cm3 de diisopropyléthylamine (2,44 mmol) et 0,48 g d'acide 3-[4-(3-tertbutoxycarbonyl-amino-propyl-tert-butoxycarbonyl-amino)-butyl-tert-butoxycarbonylamino]-acétique (FRM 375) (0,73 mmol). Au bout de 4 heures, on dilue par du dichlorométhane et on effectue un lavage à l'eau On sèche par du sulfate de magnésium et on évapore à sec sous vide Le produit obtenu est purifié par chromatographie moyenne pression dans un mélange heptane/acétate d'éthyle 6 :4. Le produit est obtenu avec un rendement de 80 % 1H RMN (CDC13) : # (ppm) 0,88 (t, 3H, J= 6,36 Hz, H-33), 1,27 (m, 14H, (CH2)25), 1,4-1,6 (m, 17H, H-2, H-17, H-14, H-17, H-37, H-40, H-41 et H-44),1,46 (m, 36H, Boc), 2,8-2,9 (m, 6H, H-15, H-16 et H-35), 3,09-3 ,33 (m, 12H, H-36, H- 38, H-39, H-42, H-43 et H-45), 3,34 (m, 1H, J= 6,71 Hz, H-la), 3,63 (m, 1H, J= 6,71 Hz, H-Ib), 3,75 (m, 1H, J= 8,97 Hz et 6,21 Hz, H-5'), 3,78 (s, 2H, CH2C6H6), 3,90 (dd, 1H, J= 6,21 Hz et J= 11,82 Hz, H-6'a), 3,97 (dd, 1H, J= 6,21 Hz et J= 11,82 Hz, H-6'b), 4,07 (s, 2H, CH2C6H6), 4,52 (dd, J= 2,91 Hz et 7,83 Hz, H-3'), 4,57 (s, 2H, CH2C6H6), 4,63 (s, 2H, CH2C6H6), 4,69 (dd, 1H, J= 2,52 Hz et 2,91 Hz, H-2'), 4,74 (1H, J= 2,52 Hz, H-l'), 4,85 (dd, 1H, J= 7,83 Hz et 8,97 Hz, H-4'), 7,35 (m, 18H, C6H6). Hz and J = 11.82 Hz, H-6'a), 3.97 (dd, 1H, J = 6.21 Hz and J = 11.82 Hz, H-6'b), 4.07 (s , 2H, CH2C6H6), 4.52 (dd, J = 2.91 Hz and 7.83 Hz, H-3 '), 4.57 (s, 2H, CH2C6H6), 4.63 (s, 2H, CH2C6H6 ), 4.69 (dd, 1H, J = 2.52 Hz and 2.91 Hz, H-2 '), 4.74 (1H, J = 2.52 Hz, H-1'), 4.85 (dd, 1H, J = 7.83 Hz and 8.97 Hz, H-4 '), 7.35 (m, 18H, C6H6).

h) Synthesis of 1- (3- (4- (3-tert-butoxycarbonyl-amino-propyl-tert-butoxycarbonyl) -butyl-tert-butoxycarbonyl-aminol-methylenecarbamoyl) -15-pentadecan-16-octadecyl-2 ', 3', 4 ', 6'-tetra-O-benzyl-BD-mannopyranose A 0.63 g (0.61 mmol) of a solution of product obtained previously in step g), in 10 cm3 of chloroform, 0.38 g of BOP (0.85 mmol), 0.425 cm3 of diisopropylethylamine (2.44 mmol) and 0.48 g of 3- [4- (3-tertbutoxycarbonylamino-propyl) tert- butoxycarbonyl-amino) -butyl-tert-butoxycarbonylamino] -acetic acid (FRM 375) (0.73 mmol). After 4 hours, the mixture is diluted with dichloromethane and washed with water. It is dried with magnesium sulphate and evaporated to dryness in vacuo. The product obtained is purified by medium pressure chromatography in a heptane / acetate mixture. ethyl 6: 4. The product is obtained with a yield of 80% 1H NMR (CDCl3): # (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.27 (m, 14H, (CH2 25), 1.4-1.6 (m, 17H, H-2, H-17, H-14, H-17, H-37, H-40, H-41 and H-44), 1 , 46 (m, 36H, Boc), 2.8-2.9 (m, 6H, H-15, H-16 and H-35), 3.09-3, 33 (m, 12H, H-36) , H-38, H-39, H-42, H-43 and H-45), 3.34 (m, 1H, J = 6.71 Hz, H-la), 3.63 (m, 1H, J = 6.71 Hz, H-Ib), 3.75 (m, 1H, J = 8.97 Hz and 6.21 Hz, H-5 '), 3.78 (s, 2H, CH 2 C 6 H 6), 3 , 90 (dd, 1H, J = 6.21 Hz and J = 11.82 Hz, H-6'a), 3.97 (dd, 1H, J = 6.21 Hz and J = 11.82 Hz, H-6'b), 4.07 (s, 2H, CH 2 C 6 H 6), 4.52 (dd, J = 2.91 Hz and 7.83 Hz, H-3 '), 4.57 (s, 2H, CH2C6H6), 4.63 (s, 2H, CH2C6H6), 4.69 (dd, 1H, J = 2.52 Hz and 2.91 Hz, H-2 '), 4.74 (1H, J = 2, 52 Hz, H-1 '), 4.85 (dd, 1H, J = 7.83 Hz and 8.97 Hz, H-4'), 7.35 (m, 18H, C6H6).

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butoxycarbonyl-amino)-butyl-tert-butoxvcarbonyl-aniinol-méthvlènecarbamoyI)-15-pentadécanvI-16-octadécvll-B-D-mannopyrannose A 0,63 g (0,38 mmol) de produit obtenu à l'étape précédente h), dans 5 cm3 de méthanol, on ajoute 10 % de palladium sur charbon (0,027 g). La solution est agitée sous pression d'hydrogène a température ambiante. Au bout de 6 heures, on filtre puis on évapore à sec sous vide. La réaction est quantitative. i) Synthesis of 1 '- [- (3- [4- (3-tert-butoxycarbonyl-amino-propyl-tert-)

butoxycarbonyl-amino) -butyl-tert-butoxycarbonyl-aniinol-methylenecarbamoyl) -15-pentadecanyl-16-octadecyl-BD-mannopyranose A 0.63 g (0.38 mmol) of the product obtained in the preceding step h), in 5 cm3 of methanol is added 10% palladium on charcoal (0.027 g). The solution is stirred under hydrogen pressure at room temperature. After 6 hours, filter and evaporate to dryness under vacuum. The reaction is quantitative.

j) Synthèse du r-f-(3-r4-(3-amino-propyl-amino)-butyI-amino1-méthylènecarbamoyi)-15-pentadécanyi-16-octadécyll-B-D-mannopyrannose (composé 1) A 0,37 g (0,28 mmol) de produit obtenu à l'étape précédente i), on ajoute 21,50 cm3 de tétrahydrofuranne (TFA) distillé. Après 1 heure, le mélange réactionnel est concentré à froid et lyophilisé. On vérifie le degré de pureté du produit en solution dans du méthanol par CLHP comme décrit dans la partie "Matériel et Méthodes". 1H NMR (CD30D): δ (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.27 (m, 14H, (CH2) 25), 1.4-1, 6 (m, 17H, H-2, H-17, H-14, H-17, H-37, H-40, H-41 and H-44), 1.46 (m, 36H, Boc), 2.8-2.9 (m, 6H, H-15, H-16 and H-35), 3.09-3.33 (m, 12H, H-36, H-38, H-39, H -42, H-43 and H-45), 3.34 (m, 1H, J = 6.71 Hz, H-la), 3.5-3.82 (m, 6H, H-2 ', H -3 ', H-4', H-5 'and H-6'), 3.63 (m, 1H, J = 6.71Hz, H-1b), 4.72 (1H, J = 2, 52 Hz, H-1 ')

j) Synthesis of rf- (3-r4- (3-aminopropylamino) butylamino-methylenecarbamoyl) -15-pentadecanyl-16-octadecyl-BD-mannopyranose (Compound 1) 0.37 g (0) 28 mmol) of product obtained in the preceding step i), 21.50 cm3 of distilled tetrahydrofuran (TFA) are added. After 1 hour, the reaction mixture is concentrated to cold and lyophilized. The degree of purity of the product in solution in methanol is checked by HPLC as described in the "Materials and Methods" section.

1H NMR (CD30D): # (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.27 (m, 14H, (CH2) 25), 1.4-1, 6 (m, 17H, H-2, H-17, H-14, H-17, H-37, H-40, H-41 and H-44), 1.46 (m, 36H, Boc), 2.8-2.9 (m, 6H, H-15, H-16 and H-35), 2.92 (m, 2H, H-45), 2.92-3.17 (m, 12H, H-36, H-38, H-39, H-42, H-43), 3.34 (m, 1H, J = 6.71 Hz, H-la), 3.5-3.82 (m.p. , 6H, H-2 ', H-3', H-4 ', H-5' and H-6 '), 3.63 (m, 1H, J = 6.71Hz, H-1b), 4 , 72 (1H, J = 2.52 Hz, H-1 ').

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méthylène-carbamoyl)-15-pentadécanyl-16-octadécyl]-a.L-6'-désoxy-manno- hexopyrannose (composé 2) a) Synthèse du 1-ol pentadécanoate de méthyle A 10 g de pentadécalactone (41,60 mmol) dans 41,60 c3mde méthanol, on ajoute 6,66 cm3 de méthylate de sodium 2N (13,31 mmol) à 0 C. Après 9 heures, on ajoute 9,24 cm3 d' acide acétique et on laisse agir pendant 15 minutes. Puis on évapore à se sous vide la solution qui est ensuite reprise par du dichlorométhane, et on effectue un lavage au bicarbonate de sodium. On sèche la phase organique obtenue par du suif de magnésium et on évapore le solvant au rotavapor La purification se fait dans mélange hexane/acétate d'éthyle 6. 4. On obtient le 1-ol-pentadécanoate de méth) avec un rendement de 80 % 1H RMN (CDCl3) : d(ppm) 1,26 (m, 12H, (CH2)10), 1,5-1,6 (m, 4H, H-2 et H-13), 2,30 (t, 2H, J= 7 60 Hz, H-14), 3,64 (t, 1H, J= 5,84 Hz, H-l), 3,67 (s, 3H, H-16).

b) Synthèse du V-hentadécan-15-oate de méthyll-2',3',4'-tri-0-acétyl- cc-L-6'- désoxy-manno-hexopyrannose A O C, on ajoute 2,49 en? de chlorure d'étain (21,30 mmol) à 3,55 g de rhamnose peracétylé (10,65 mmol) dans 27 cm de dichlorométhane pendant 30 minutes. Puis on additionne 3,48 g de 1-ol pentadécanoate de méthyle précédemment obtenu (12,7 mmol). Après 2 heures, le mélange réactionnel est dilué par de l'éther éthylique et on verse dans une solution de phosphate de sodium (N2PO4) Les phases aqueuses sont extraites avec du diéthyléther et les phases organiques sont successivement lavées p@ une solution de carbonate de sodium, de la saumure, puis séchées par du sulfate c magnésium Après évaporation à sec sous vide, on purifie par chromatograph moyenne pression dans un mélange hepta[alpha]ne/[alpha]te[alpha]te dëthyle 7#3. Le produit est obtenu avec un rendement de 60 %. Example 2 Synthesis of 1 '- [- (3- [4- (3-aminopropylamino) butylamino] -

methylene-carbamoyl) -15-pentadecanyl-16-octadecyl] -α-6'-deoxy-manno-hexopyranose (compound 2) a) Synthesis of methyl 1-ol pentadecanoate to 10 g of pentadecalactone (41.60 mmol) in 41.60 cm3 of methanol is added 6,66 cm3 of 2N sodium methylate (13.31 mmol) at 0 ° C. After 9 hours, 9.24 cm3 of acetic acid are added and the mixture is left to act for 15 minutes. Then the solution is evaporated under vacuum, which is then taken up in dichloromethane, and a washing with sodium bicarbonate is carried out. The organic phase obtained is dried with magnesium tallow and the solvent is evaporated off in a rotary evaporator. The purification is carried out in a hexane / ethyl acetate mixture. 4. Methyl 1-ol-pentadecanoate is obtained in a yield of 80.degree. 1 H NMR (CDCl 3): d (ppm) 1.26 (m, 12H, (CH 2) 10), 1.5-1.6 (m, 4H, H-2 and H-13), 2.30 ( t, 2H, J = 760 Hz, H-14), 3.64 (t, 1H, J = 5.84 Hz, H1), 3.67 (s, 3H, H-16).

b) Synthesis of Methyl-2 ', 3', 4'-tri-O-acetyl-α-L-6'-deoxy-manno-hexopyranose-5-o-hentadecan-15-oate AOC is added 2.49 in. of tin chloride (21.30 mmol) to 3.55 g of peracetylated rhamnose (10.65 mmol) in 27 cm of dichloromethane for 30 minutes. 3.68 g of previously obtained methyl 1-ol pentadecanoate (12.7 mmol) are then added. After 2 hours, the reaction mixture is diluted with ethyl ether and poured into a solution of sodium phosphate (N2PO4). The aqueous phases are extracted with diethyl ether and the organic phases are successively washed with a solution of sodium carbonate. Sodium, brine and then dried with magnesium sulfate. After evaporation to dryness in vacuo, the mixture is purified by medium pressure chromatography in a mixture hepta [alpha] n / [alpha] te [alpha] teethyl 7 # 3. The product is obtained with a yield of 60%.

1 H NMR (CDCl 3): d (ppm) 1.20 (d, 3H, J = 6.45 Hz, H-6 '), 1.26 (m, 12H, (CH 2) 10), 1.59 (m , 4H, H-2 and H-13), 1.98,2.04 and 2.16 (s, 3H, OCOCH3), 2.29 (t,

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 2H, J = 7.62Hz, H-14), 3.40 (m, 1H, J = 6.71Hz, H-1a), 3.66 (m, 1H, J = 6.71Hz, Hlb). ), 3.67 (s, 3H, H-16), 3.88 (m, 1H, J = 6.45 Hz and 9.97 Hz, H-5 '), 4.70 (d, 1H, J); = 1.72 Hz, H-1 '), 5.06 (dd, 1H, J = 9.97 Hz and 9.97 Hz, H-4'), 5.22 (dd, 1H, J = 1, 72 Hz and 3.52 Hz, H-2 '), 5.30 (dd, 1H, J = 3.52 Hz and 9.97 Hz, H-3'). c) Synthesis of Methyl] - [alpha] -L-6'-deoxy-manno-hexopyranose r-fpentadecan-15-oate 5.08 g of the product obtained in step b) (9.34 mmol) is treated dissolved in 20 cm3 of methanol with 9.34 ml of 2N sodium methylate (18.68 mmol). When the reaction is complete, the reaction mixture is neutralized with Amberlite IR120, filtered and evaporated to dryness in vacuo.

d) Synthèse du l'-fpentadécan-15-oate de méthvll-2',3',4'-tri-O-benzyl-a-L-6'- désoxy-manno-hexopyrannose A 2,09 g (5,00 mmol) de produit obtenu à l'étape précédente c), dans 30 cm3 de diméthylformamide (DMF) anhydre, on ajoute successivement 3,32 g d'iodure de potassium (20,00 mmol), 0,80 g d'hydrure de sodium 60 % (20,00 mmol) et 2,38 cm3 du bromure de benzyle (20,00 mmol). Après 12 heures, on ajoute 20 cm3 d'une solution saturée de chlorure d'ammonium et on laisse agir pendant 10 minutes. Puis, on dilue avec de l'eau et on extrait la phase organique par de l'acétate d'éthyle. Celle-ci est ensuite lavée avec de l'eau et de la saumure, avant d'être finalement séchée par du sulfate de magnésium. 1H NMR (CDCl3): δ (ppm) 1.20 (d, 3H, J = 6.45Hz, H-6 '), 1.26 (m, 12H, (CH2) 10), 1.59 (m). , 4H, H-2 and H-13), 2.29 (t, 2H, J = 7.62 Hz, H-14), 3.40 (m, 1H, J = 6.71 Hz, H-la ), 3.66 (m, 1H, J = 6.71Hz, H-1b), 3.67 (s, 3H, H-16), 3.6-3.9 (m, 4H, H-2) ', H-3', H-4 'and H-5'), 4.70 (d, 1H, J = 1.72 Hz, H-1 ').

d) Synthesis of Methyl-2 ', 3', 4'-tri-O-benzyl-α-L-6'-deoxy-manno-hexopyranose 1-fpentadecan-15-oate A 2.09 g (5.00 mmol) ) of product obtained in the preceding step c), in 30 cm3 of anhydrous dimethylformamide (DMF), 3.32 g of potassium iodide (20.00 mmol), 0.80 g of sodium hydride are successively added; 60% (20.00 mmol) and 2.38 cc of benzyl bromide (20.00 mmol). After 12 hours, 20 cm3 of saturated ammonium chloride solution are added and the mixture is allowed to act for 10 minutes. Then, it is diluted with water and the organic phase is extracted with ethyl acetate. This is then washed with water and brine before being finally dried with magnesium sulfate.

On the other hand, an additional washing with a saturated solution of sodium thiosulfate is carried out in order to eliminate the iodide ions. It is evaporated to dryness in vacuo and the resulting oil is purified in heptane / ethyl acetate 9: 1. The product is obtained with a yield of 60%.

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1 H NMR (CDCl 3): δ (ppm) 1.28 (m, 14H, (CH 2) 10), 1.33 (d, 3H, J = 6.21 Hz, H-6 '), 1.59 (m.p. m, 4H, H-2 and H-13), 2.31 (t, 2H, J = 7.62 Hz, H-14), 3.40 (m, 1H, J = 6.71 Hz, H-13). la), 3.65 (s, 2H, CH2C6H6), 3.66 (m, 1H, J = 6.71 Hz, H-1b), 3.67 (s, 3H, H-16), 3.68 (m, 1H, J = 9.5 Hz and 6.21 Hz, H-5 '), 3.99 (s, 2H, CH 2 C 6 H 6), 4.02 (dd, 1H, J = 8.96 Hz and 9 , 5Hz, H-4 '), 4.32 (s, 2H, CH2C6H6), 4.57 (dd, 1H, J = 2.01Hz and 3.02Hz, H-2'), 4.73 (1H, J = 2.01 Hz, H-1 '), 4.82 (dd, J = 3.02 Hz and 8.96 Hz, H-3'), 7.35 (m, 18H, C6H6) . e) Synthesis of 5α-pentaecan-15-o-piper-2 ', 3', 4'-tri-O-benzyl-α-L-6'-δoxy-mannohexopyranose A 0.50 g (0.73 mmol) ) of a product solution obtained in the preceding step d), in 7 cm3 of methanol, 4.68 ml of 25% sodium hydroxide are added. The reaction mixture is refluxed for 30 minutes. Then, the cold mixture is neutralized with a 5% hydrochloric acid solution. The organic phase is extracted with ethyl acetate and evaporated to dryness in vacuo. Purification is in heptane / ethyl acetate 4: 6. product is obtained with a yield of 72%.

f1 Synthèse 8u r-[pentaSécan-15-carbamovl-16-octa5écvll-2%3\4'-tri-Q-benzyI- [alpha]-L-6'-#ésoxy-manno-hexopyrannose A 0,70 g (1,04 mmol) d'une solution de produit précédemment obtenu à l'étape e), dans 13 cm3 de chloroforme, on ajoute successivement 0,69 g de BOP (1,56 mmol), 0,72 cm3 de diisopropyléthylamine (4,16 mmol) et 0,34 g d'octadécylamine (1,25 mmol) Lorsque la réaction est terminée, on dilue par du dichlorométhane, on effectue un lavage à l'eau, on sèche sur sulfate de magnésium, et on évapore à sec 1 H NMR (CDCl 3): δ (ppm) 1.28 (m, 14H, (CH 2) 10), 1.33 (d, 3H, J = 6.21 Hz, H-6 '), 1.59 ( m, 4H, H-2 and H-13), 2.34 (t, 2H, J = 7.62 Hz, H-14), 3.40 (m, 1H, J = 6.71 Hz, H- la), 3.65 (s, 2H, CH2C6H6), 3.66 (m, 1H, J = 6.71 Hz, H-1b), 3.68 (m, 1H, J = 9.5 Hz and 6). , 21 Hz, H-5 '), 3.99 (s, 2H, CH2C6H6), 4.02 (dd, 1H, J = 8.96 Hz and 9.5 Hz, H-4'), 4. 32 (s, 2H, CH 2 C 6 H 6), 4.57 (dd, 1H, J = 2.01 Hz and 3.02 Hz, H-2 '), 4.73 (1H, J = 2.01 Hz, H-1). 4.82 (dd, J = 3.02 Hz and 8.96 Hz, H-3 '), 7.35 (m, 18H, C6H6).

Synthesis 8 R- [pentaSecan-15-carbamoyl-16-octa5ecvll-2% 3 \ 4'-tri-Q-benzyl] [alpha] -L-6 '- hexoxymethylhexopyranose A 0.70 g ( 1.04 mmol) of a product solution previously obtained in stage e), in 13 cm 3 of chloroform, 0.69 g of BOP (1.56 mmol), 0.72 cm 3 of diisopropylethylamine (4%) are added successively. 16 mmol) and 0.34 g of octadecylamine (1.25 mmol). When the reaction is complete, the mixture is diluted with dichloromethane, washed with water, dried over magnesium sulphate and evaporated at room temperature. dry

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e) Synthèse du r-fpentadecan-15-amino-16-octadecvn-2\3>,4>-tri-0-benzyl-a- L-6'-désoxy-manno-hexopyrannose A 0,81 g (0,86 mmol) de produit obtenu à l'étape précédente f), dans 15 cm3 de tétrahydrofuranne (THF) anhydre, on ajoute 0,065 g d'hydrure de lithium, aluminium AlLiH4 (1,72 mmol), et on chauffe à reflux pendant 10 heures Puis, le mélange réactionnel est refroidi dans de un bain de glace et on ajoute 65 (il d'eau, puis 130 l de soude 2N au bout de 10 minutes, et enfin à nouveau 65 {il d'eau après 10 minutes On filtre et on évapore à sec sous vide La purification se fait dans un mélange dichlorométhane/méthanol/ammoniac 28% 9 2 0,5 Le produit est obtenu avec un rendement de 93 %. under vacuum. The product obtained is purified by medium pressure chromatography in a heptane / ethyl acetate mixture 6: 4. The product is obtained with a yield of 84% 1 H NMR (CDCl 3) δ (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.27 (m, 14H, (CH 2 25), 1.33 (d, 3H, J = 6.21 Hz, H-6 '), 1.47 (m, 4H, H-2 and H-17), 1.58 (m, 2H, H-13), 2.13 (t, 2H, J = 7.92 Hz, H-14), 3.23 (m, 2H, H-16), 3.40 (m, 1H, J = 6, 71Hz, H-1α), 3.65 (s, 2H, CH 2 C 6 H 6), 3.66 (m, 1H, J = 6.71Hz, H-1b), 3.68 (m, 1H, J = 9; , 5 Hz and 6.21 Hz, H-5 '), 3.99 (s, 2H, CH 2 C 6 H 6), 4.02 (dd, 1H, J = 8.96 Hz and 9.5 Hz, H-4' ), 4.32 (s, 2H, CH2C6H6), 4.57 (dd, 1H, J = 2.01 Hz and 3.02 Hz, H-2 '), 4.73 (1H, J = 2.01 Hz). , H1 '), 4.82 (dd, J = 3.02 Hz and 8.96 Hz, H-3'), 5.37 (band, 1H, HNCO), 7.35 (m, 18H, C6H6)

e) Synthesis of r-pentadecan-15-amino-16-octadecyl-2β, 4β-tri-O-benzyl-α-L-6'-deoxy-mannohexopyranose A 0.81 g (0); 86 mmol) of product obtained in the preceding step f), in 15 cm3 of anhydrous tetrahydrofuran (THF), 0.065 g of lithium aluminum hydride AlLiH4 (1.72 mmol) are added and the mixture is refluxed for 10 minutes. Then, the reaction mixture is cooled in an ice bath and 65 ml of water are added, then 130 l of 2N sodium hydroxide after 10 minutes, and finally 65 ml of water again after 10 minutes. The product is obtained in a yield of 93%.

1 H NMR (CDCl 3) δ (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.27 (m, 14H, (CH 2) 25), 1.33 (d, 3H, J = 6.21 Hz, H-6 '), 1.4-1.6 (m, 9H, H-2, H-17, H-14, H-17 and NH), 2.57 (t , 4H, J = 7.92 Hz, H-15 and H-16), 3.40 (m, 1H, J = 6.71 Hz, H-la), 3.65 (s, 2H, CH 2 C 6 H 6), 3.66 (m, 1H, J = 6.71 Hz, H-1 b), 3.68 (m, 1H, J = 9.5 Hz and 6.21 Hz, H-5 '), 3.99 ( s, 2H, CH 2 C 6 H 6), 4.02 (dd, 1H, J = 8.96 Hz and 9.5 Hz, H-4 '), 42 (s, 2H, CH 2 C 6 H 6), 4.57 (dd, 1H, J). = 2.01 Hz and 3.02 Hz, H-2 '), 4.73 (1H, J = 2.01 Hz, H-1'), 4.82 (dd, J = 3.02 Hz and 8 , 96 Hz, H-3 '), 7.35 (m, 18H, C6H6)

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butoxycarbonyl-amino)-butyl-tert-butoxycarbonyl-aminol-méthvlènecarbamoyl)-15-pentadécanyl-16-octadécyll-2',3',4'-tri-O-benzyl-a-L-6'-désoxv- manno-hexopyrannose A 0,78 g (0,86 mmol) d'une solution de produit obtenu à l'étape précédente g), en solution dans 7 cm3 de chloroforme, on ajoute successivement 0,53 g de BOP (1,20 mmol), 0,30 cm3 de diisopropyléthylamine (1,72 mmol) et 0,62 g de FRM 375 (0,95 mmol) Après 4 heures, on dilue avec du dichlorométhane, on effectue un lavage à l'eau, on sèche sur sulfate de magnésium, et on évapore à sec sous vide. Le produit obtenu est purifié par chromatographie "flash" dans un mélange heptane/acétate d'éthyle 6 4 Le produit est obtenu avec un rendement de 72 %. h) Synthesis of 1 '- [- (3- [4- (3-tert-butoxycarbonyl-amino-propyl-tert-)

butoxycarbonylamino) butyl-tert-butoxycarbonylamino-methylenecarbamoyl) -15-pentadecanyl-16-octadecyl-2 ', 3', 4'-tri-O-benzyl-α-6'-deoxyman-hexopyranose A 0.78 g (0.86 mmol) of a solution of product obtained in the preceding step g), dissolved in 7 cm3 of chloroform, are added successively 0.53 g of BOP (1.20 mmol), 30 cm3 of diisopropylethylamine (1.72 mmol) and 0.62 g of FRM 375 (0.95 mmol). After 4 hours, the mixture is diluted with dichloromethane, washed with water and dried over magnesium sulphate. and evaporated to dryness under vacuum. The product obtained is purified by flash chromatography in a heptane / ethyl acetate mixture. The product is obtained with a yield of 72%.

butoxycarbonyl-amino)-butyl-tert-butoxycarbonyl-aminol-méthylène-carbamoyl)-15-pentadécanyl-16-octadécyll-a-L-6'-désoxy-manno-hexopyrannose A 0,74 g (0,48 mmol) de produit obtenu à l'étape précédente h), en solution dans 10 cm3 de méthanol, on ajoute 10 % (0,034 g) de palladium sur charbon La solution est agité sous pression d'hydrogène a température ambiante. Après 4 heures, on filtre puis on évapore à sec sous vide. La réaction est quantitative. 1 H NMR (CDCl 3) δ (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.27 (m, 14H, (CH 2) 25), 1.33 (d, 3H, J = 6.21 Hz, H-6 '), 1.4-1.6 (m, 17H, H-2, H-17, H-14, H-17, H-37, H-40 , H-41 and H-44), 1.46 (m, 36H, Boc), 2.8-2.9 (m, 6H, H-15, H-16 and H-35), 3.09- 3.33 (m, 12H, H-36, H-38, H-39, H-42, H-43 and H-45), 3.40 (m, 1H, J = 6.71 Hz, H- la), 3.65 (s, 2H, CH2C6H6), 3.66 (m, 1H, J = 6.71 Hz, H-1b), 3.68 (m, 1H, J = 9.5 Hz and 6). , 21 Hz, H-5 '), 3.99 (s, 2H, CH 2 C 6 H 6), 4.02 (dd, 1H, J = 8.96 Hz and 9.5 Hz, H-4'), 4 32 ( s, 2H, CH 2 C 6 H 6), 4.57 (dd, 1H, J = 2.01 Hz and 3.02 Hz, H-2 '), 4.73 (1H, J = 2.01 Hz, H-1). ), 4.82 (dd, J = 3.02 Hz and 8.96 Hz, H-3 '), 7.35 (m, 18H, C6H6). i) Synthesis of 1 '- [- (3- [4- (3-tert-butoxycarbonyl-amino-propyl-tert-)

butoxycarbonyl-amino) -butyl-tert-butoxycarbonyl-aminol-methylene-carbamoyl-15-pentadecanyl-16-octadecyl-α-6'-deoxy-manno-hexopyranose A 0.74 g (0.48 mmol) of the product obtained in the preceding step h), dissolved in 10 cm3 of methanol, 10% (0.034 g) of palladium on charcoal is added. The solution is stirred under hydrogen pressure at room temperature. After 4 hours, filter and evaporate to dryness under vacuum. The reaction is quantitative.

1 H NMR (CD 3 OD) δ (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.20 (d, 3H, J = 6.45 Hz, H-6 ') , 1.27 (m, 14H, (CH 2) 25), 1.4-1.6 (m, 17H, H-2, H-17, H-14, H-17, H-37, H-40 , H-41 and H-44), 1.46 (m, 36H, Boc), 2.8-2.9 (m, 6H, H-15, H-16 and H-35), 3.09-

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n Synthèse du l'-[-(3-[4-(3-amino-propyl-amino)-butvl-aminol-méthylenecarbamoyl)-15-pentadécanyl-16-octadécvIl-a-L-6'-désoxy-manno-hexopvran- nose (composé 2) A 0,40 g (0,31mmol) de produit obtenu à l'étape précédente i), on ajoute 24 cm3 de tétrahydrofuranne (TFA) distillé. Après 1 heure, le mélange réactionnel est évaporé sous vide à froid et à sec, puis est lyophilisé. On vérifie le dégré de pureté du produit en solution dans du méthanol par CLHP. 3.33 (m, 12H, H-36, H-38, H-39, H-42, H-43 and H-45), 3.40 (m, 1H, J = 6.71 Hz, H1a). , 3.66 (m, 1H, J = 6.71 Hz, H-1b), 3.6-3.9 (m, 4H, H-2 ', H-3', H-4 'and H- 5 '), 4.73 (1H, J = 2.11 Hz, H-1')

Synthesis of - [- (3- [4- (3-aminopropylamino) -butyl-aminol-methylenecarbamoyl) -15-pentadecanyl-16-octadecyl-α-6'-deoxy-manno-hexopyran nose (compound 2) To 0.40 g (0.31 mmol) of product obtained in the preceding step i), 24 cm3 of distilled tetrahydrofuran (TFA) are added. After 1 hour, the reaction mixture is evaporated under vacuum to dryness and dry, and is lyophilized. The degree of purity of the product in solution in methanol is checked by HPLC.

1H NMR (CD30D). # (ppm) 0.88 (t, 3H, J = 6.36 Hz, H-33), 1.20 (d, 3H, J = 6.45 Hz, H-6 '), 1.27 (m , 14H, (CH 2) 25), 1.4-1.6 (m, 17H, H-2, H-17, H-14, H-17, H-37, H-40, H-41 and H -44), 2.8-2.9 (m, 6H, H-15, H-16 and H-35), 2.92 (m, 2H, H-45), 2.92-3.17 ( m, 12H, H-36, H-38, H-39, H-42, H-43), 3.40 (m, 1H, J = 6.71 Hz, H-la), 3.66 (m.p. , 1H, J = 6.71 Hz, H-1b), 3.6-3.9 (m, 4H, H-2 ', H-3', H-4 'and H-5'), 4, 73 (1H, J = 2.01 Hz, H-1 ').

EXAMPLE 3 Preparation of Transfer Agent / Nucleic Acid Complexes with Compound 2 and Measurement of Their Size This example illustrates the preparation of complexes between a transfer agent according to the invention and a nucleic acid, their size then being measured.

The glycolipid used in this example and in the following examples is compound 2, dissolved in chloroform, at a concentration of 10 mg / ml. In some cases, a neutral co-lipid, cholesterol or DOPE was premixed with compound 2.

The lipid solution is prepared as follows: a sample of the desired quantity is taken, the solvent is evaporated under an argon stream and allowed to dry for

<Desc / Clms Page number 35>

 1 hour. Then, the lipid is rehydrated with a solution containing dextrose 5% and 10 mM sodium chloride overnight at 4 C. The following day, the lipid solutions are heated at 60 C for 5 minutes and then sonicated for 1 hour. minute. The operation is repeated until the size of the lipid particles is stable.

The DNA used is the plasmid pXL3031 (FIG. 1) in solution in a mixture of 5% dextrose and 10 mM sodium chloride at a concentration of 0.5 mg / ml or 1.0 mg / ml. This plasmid contains luc gene coding for luciferase under the control of the cytomegalovirus P / E CMV promoter. Its size is 3671 bp. The scheme of this plasmid is shown in FIG. 1. The plasmid pXL3031 was purified according to the methods described in the patent application WO 97/35002.

Compound 2 / DNA complexes are prepared by rapidly mixing appropriate volumes of plasmid DNA solution and Compound 2 (at the desired charge ratio) at room temperature. The amount of transfectant varies between 0.25 nmol / g DNA and 12 nmol / μg DNA.

The size of the complexes was analyzed by measuring the hydrodynamic diameter by Dynamic Laser Light Scattering using a Coulter N4Plus device. Samples are diluted 20-fold in a solution containing 5% dextrose and 20 mM sodium chloride to avoid multiple scattering.

At a ratio of 3 nmol of lipid / μg of DNA, the following results were obtained:

<Tb>
<tb> Size <SEP> in <SEP> nm
<tb> Micelles <SEP> 130 <SEP> nm
<tb> formulation <SEP> with <SEP> of <SEP> Cholesterol <SEP> 153 <SEP> nm
<tb> Formulation <SEP> with <SEP> of <SEP><SEP> DOPE <SEP> 137 <SEP> nm
<Tb>
The term "micelles" indicates that compound 2 has been used alone, that is, without addition of neutral co-lipid, and thus forms a micellar solution.

<Desc / Clms Page number 36>

 This table shows that the complexes obtained have a size between about 130 nm and 150 nm, which is compatible with a pharmaceutical use, particularly in injection.

Example 4 Behavior of Complexes Formed from Compound 2 at Different Charge Ratios This example illustrates the behavior of the transfer agent complexes according to the invention / nucleic acid when the charge ratio is varied. The impact of adding a co-lipid (cholesterol or DOPE) is also illustrated.

Conventionally, three physicochemical phases are distinguished when increasing the transfer agent / DNA charge ratio (B. Pitard et al., Virus-sized self-assembly lamellar complexes between plasmid DNA and cationic micelles promote gene transfer, PNAS, Vol 94, pp. 14412-14417, 1997). These three phases determine the therapeutic potential of the transfer agent.

 At a low charge ratio, the DNA is not saturated with the transfer agent. There is still non-complexed DNA, and the complexes are globally negatively charged and small in size. This phase, stable, is called "Phase A".

The fact that the DNA is not completely saturated with the transfer agent means that the DNA is not completely protected. DNA can therefore be subject to degradation by enzymes. Moreover, the complexes being generally negative, the passage of the cell membrane is difficult. For these reasons, the nucleolipidic complexes of phase A are relatively inactive
At an intermediate charge ratio, the DNA is completely saturated with the transfer agent, and the complexes are generally neutral or slightly positive. This phase is unstable because the ionic repulsions are minimal and a phenomenon of "crosslinking" can occur. The particle size is well above the detection limit by dynamic light scattering (much greater than 3 m). This unstable phase is called "phase B". Such a complex size is not suitable for injection uses, although this does not mean that

<Desc / Clms Page number 37>

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"UIlIP";)};)) auitn 1114 \ "Ul;) U411;) your puttac D. Il;) bulit JGUIGIIIGIII suas a lunnutanon that is not suitable for their injection for a pharmaceutical purpose.

 At a higher charge ratio, the DNA is over-saturated with the transfer agent, and the complexes are generally positive. Because of strong repulsions between positive charges, this phase is stable. It is referred to as "phase C".

Unlike phase A, the complexes obtained are in such a form that the DNA is very well protected against enzymes, and their overall positive charge facilitates the passage of the cell membrane of anionic nature. The complexes of phase C are therefore particularly suitable for use for the transfer of nucleic acids into cells.

These 3 zones A, B and C have also been updated with compound 2 according to the invention as transfer agent:

<Tb>
<tb><SEP> Ratio of <SEP> 0.25 <SEP>#<SEP> 0.5 <SEP>#<SEP> 0.75 <SEP>#<SEP> 1 <SEP>#<SEP> 1 , 5 <SEP>#<SEP> 2 <SEP>#<SEP> 3 <SEP>#<SEP> 4 <SEP>#<SEP> 6 <SEP> 8 <SEP> 10 <SEP> 12 <SEP>
<tb> charges
<tb> Micelles <SEP> A <SEP> A <SEP> B <SEP> B <SEP> B <SEP> B <SEP> C <SEP> C <SEP> C <SEP> C <SEP> C <SEP > C
<tb> + <SEP> Cholesterol <SEP> A <SEP> A <SEP> B <SEP> B <SEP> B <SEP> C <SEP> C <SEP> C <SEP> C <SEP> C <SEP > C <SEP> C
<tb> + <SEP> DOPE <SEP> A <SEP> A <SEP> B <SEP> B <SEP> B <SEP> C <SEP> C <SEP> C <SEP> C <SEP> C <SEP > C <SEP> C
<Tb>
As shown in the table above, area B, which is the area of instability, is particularly small and is at very low load ratios. Zone C starts from 2 nmol of lipid / μg of DNA when compound 2 is used together with a co-lipid (Cholesterol or DOPE), and from 3 nmol of lipid / g of DNA when the compound is used only. As has been stated previously, it is in this area that it is particularly advantageous to place oneself for a pharmaceutical use.

Thus, compound 2 is a particularly advantageous transfer agent because it is stable at low charge ratios, which makes it possible to form stable complexes

<Desc / Clms Page number 38>

 with small amounts of glycolipids, with the consequent beneficial consequences in terms of toxicity.

 Example 5 Use of Compound 2 for the In Vitro Transfer of DNA This example illustrates the ability of the transfer agents according to the invention to transfect DNA in cells in vitro, at different charge ratios, in the absence and in the presence of a neutral co-lipid (cholesterol or DOPE).

 24-well microplates are inoculated with 60000 HeLa cells per well, and are grown overnight. The number of cells after one night, and therefore at the time of transfection, is 100,000 cells per well.

Each well is brought into contact with the complexes formed with compound 2 and containing 1 g of plasmid DNA in 0.5 ml of DMEM culture medium (Gibco / BRL without serum. The cells are incubated at 37 ° C. for 5 hours. The medium containing the complexes is then removed and replaced with a DMEM culture medium and 10% fetal calf serum.The cells are then cultured again for 24 hours. a luciferase test kit (Promega) and a Dynex MLX luminometer.

The results obtained are indicated on the histogram of FIG. 2. The efficiency of the transfer is represented by the expression of luciferase in μg / well. It is found that the maximum transfection is approximately 500 μg / well. In conclusion, this example clearly shows that it is possible to use the compound 2 according to the invention to form complexes capable of promoting the transfer of DNA into the cells. cells in vitro.

 Example 6: Use of Compound 2 for In Vivo Transfer of DNA This example illustrates the ability of the transfer agents according to the invention to transfect DNA into cells in vivo.

In vivo gene transfer was performed on Balb / C mice by intratracheal, intravenous and intramuscular administration

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 In the case of intramuscular injections, each mouse received 30 l of formulation containing 15 g of plasmid DNA in the anterior tibia muscle. The tissues are recovered 7 days after the injection, they are frozen and stored at -80 C while waiting for the luciferase activity tests.

In the case of intravenous injections, each mouse received 200 l of formulation containing 50 g of plasmid DNA. The tissues are recovered 24 hours after the injection, then are frozen and stored in the same way as before.

Figure 3 illustrates the activity of complexes formed with compound 2 for intramuscular gene transfer in vivo. These results clearly show that the formation of complexes with the compound 2 according to the invention and DNA makes it possible to promote the transfer of said DNA into the cells in vivo.

In the same way, any transfer agent as defined in the present invention can be used to promote the transfer of DNA into the cells.

Claims (25)

 1. Nucleic acid transfer agents characterized in that they comprise a hydrophobic part bonded to at least two hydrophilic parts, at least one of which is a sugar. Nucleic acid transfer agents according to claim 1 of general formula (I):

 for which: R represents a polycation; Z represents a hydrogen atom or a fluorine atom, the different Zs being independent of one another; and x and y, independently of one another, represent an integer; between 10 and 22 inclusive, and - X and Y, independently of each other, represent a hydrogen atom, an OAlk group, Alk representing a straight or branched alkyl containing 1 to 4 carbon atoms, a group amino, a sugar, a peptide, an oligonucleotide or a marker, at least one of the substituents X and Y being a sugar, where appropriate in their isomeric forms, as well as mixtures thereof, or their salts when they exist. 3. Nucleic acid transfer agents according to claim 2 characterized in that the one or more sugars are mono-, oligo- or polysaccharide molecules which interact with cellular receptors. <Desc / Clms Page number 41>  4. Nucleic acid transfer agents according to claim 3 characterized in that said one or more sugars are glucose, mannose, rhamnose, galactose, fructose, maltose, lactose, sucrose, sucrose, fucose, cellobiose, allose, laminarabiose, gentiobiose, sophorose, or melibiose. 5. Nucleic acid transfer agents according to claim 3 characterized in that said one or more sugars are complex sugars. 6. Nucleic acid transfer agents according to claim 2 characterized in that said oligonucleotide is any chain containing one or more nucleotides, deoxynucleotides, ribonucleotides and / or deoxyribonucleotides, optionally coupled to one or more molecules having distinct properties. 7. Nucleic acid transfer agents according to claim 2 characterized in that said peptide is any chain containing one or more amino acids linked together by peptide bonds, optionally substituted by one or more aliphatic groups which can be saturated or unsaturated, and linear, branched or cyclic. 8. Nucleic acid transfer agents according to claim 2 characterized in that said marker is any agent for identification by analysis techniques such as fluorescence spectrometry, infrared spectrometry or nuclear magnetic resonance. 9. Nucleic acid transfer agents according to claim 2 characterized in that the polycation R is a linear or branched hydrophilic polycationic molecule capable of associating with the nucleic acid. 10. Nucleic acid transfer agents according to claim 2 or 9 characterized in that the polycation R represents a polyamine of general formula (II): <Desc / Clms Page number 42>  in which: R1, R2 and R3 independently of one another represent a hydrogen atom or a group (CH2) qNR'R "with q an integer ranging between 1, 2,3, 4,5 and 6 inclusive; in an independent manner between the different groups R 1, R 2 and R 3, R 'and R "represent, independently of one another, a hydrogen atom or a group (CH 2) qNH 2 with q defined as above, and m, n and p independently of one another represent an integer ranging from 0.1, 2.3, 4.5 and 6 inclusive, with when n is greater than 1, m being able to take different values and R3 having meanings different in the general formula (II).

11. Nucleic acid transfer agent according to claim 10, characterized in that the polycation R represents spermine or spermidine. 12. Nucleic acid transfer agents according to claim 2 characterized in that, in the general formula (I), Y represents a hydrogen atom and X represents a sugar. 13. Nucleic acid transfer agents according to claim 11, characterized in that in addition the polycation R represents a polyamine of the general formula (II) defined in claim 10. 14. Transfer agent according to claim 1 of formula: <Desc / Clms Page number 43>

 15. Transfer agent according to claim 1 of formula:

 16. A composition characterized in that it contains a nucleic acid transfer agent as defined in the preceding claims and a nucleic acid. 17. Composition according to claim 16 characterized in that the nucleic acid is a deoxyribonucleic acid or a ribonucleic acid. Composition according to claim 16 or 17 characterized in that said nucleic acid comprises one or more genes of therapeutic interest under the control of regulatory sequences. 19. Composition according to claims 16 to 18 characterized in that said nucleic acid is an antisense gene or sequence. Composition according to Claim 16, characterized in that it additionally contains one or more adjuvants. 21. Composition according to claim 20, characterized in that the adjuvant is one or more neutral lipids. Composition according to Claim 21, characterized in that the neutral lipids are lipids with two fatty chains. <Desc / Clms Page number 44>  23. A composition according to claims 21 and 22 characterized in that the neutral lipids are natural or synthetic lipids, zwitterionic or lacking ionic charge under physiological conditions, chosen for example from dioleoyl p hosphatidylethanol amine (DOPE), oleylpalmitoylphosphatidylethanolamine (POPE), di-stearoyl, -palmitoyl, -mirystoylphosphatidylethanolamines and their N-methylated derivatives 1 to 3 times, phosphatidylglycerols, diacylglycerols, glycosyldiacylglycerols, cerebrosides (such as in particular galactocerebrosides), sphingolipids (such as especially sphingomyelins) or asialogangliosides (such as in particular asialoGM1 and GM2) 24. Composition according to claim 20, characterized in that said adjuvant is a compound directly or indirectly involved in the condensation of the nucleic acid. Composition according to Claim 24, characterized in that the said adjuvant is derived in whole or in part from a protamine, a histone, or a nucleolin and / or from one of their derivatives, or consists, in all or part of peptide units (KTPKKAKKP) and / or (ATPAKKAA), the number of units may vary between 2 and 10, and can be repeated continuously or not 26 Composition according to claims 16 to 25 characterized in that it comprises a pharmaceutically acceptable vehicle for an injectable formulation 27. A composition according to claims 16 to 25, characterized in that it comprises a pharmaceutically acceptable vehicle for application to the skin and / or the mucous membranes. Transfer according to one of claims 1 to 15 for the manufacture of a medicament 29 Use of a transfer agent according to one of claims 1 to 15 for the preparation of a medicament for treating diseases resulting from a deficiency in a protein or nucleic product <Desc / Clms Page number 45>  30. A method for transferring nucleic acids in vitro or ex vivo into cells, characterized in that it comprises the following steps: (1) contacting the nucleic acid with a transfer agent as defined in the claims 1 to 14, to form a complex, and (2) contacting the cells with the complex formed in (1) 31In vitro or ex vivo nucleic acid transfer method in cells according to claim 30 characterized in that said transfer agent and / or said nucleic acid are premixed with one or more adjuvant (s) as defined above.